To compare the corneal asphericity and higher-order aberrations (HOAs) of femtosecond laser-assisted in situ keratomileusis (FS-LASIK) with Smart Pulse Technology (SPT) assisted transepithelial photorefractive keratectomy (Trans-PRK) for myopia and myopic astigmatism correction. Q value) of the anterior corneal surface, index of surface variance (ISV), corneal higher-order aberrations (HOAs), vertical coma (Z3−1), horizontal coma (Z31), and spherical aberration (Z40) over a 6 mm diameter central corneal zone diameter were evaluated preoperatively and 1, 3, and 6 months postoperatively.  This prospective study analyzed 88 eyes of 44 patients treated with FS-LASIK and 64 eyes of 32 patients treated with Trans-PRK. All eyes had low to moderate myopia with or without astigmatism  <−6.00 diopters). The uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), SE, asphericity (P > 0.05). After FS-LASIK and Trans-PRK, the Q values in the 6, 7, 8, and 9 mm zones and ISV of the anterior corneal surface significantly increased (P < 0.001). At 1, 3, and 6 months after surgery, corneal HOA, Z3−1, Z31, and Z40 in both groups were significantly increased compared with those before surgery, with statistically significant differences (P < 0.001). At 1, 3, and 6 months after surgery, the Z3−1 of the Trans-PRK group was significantly lower than that of the FS-LASIK group (P < 0.001). ΔHOA and ΔZ40 were dramatically correlated with the ΔQ value for both FS-LASIK and Trans-PRK procedures. The ΔQ was significantly correlated with the preoperative SE, AD, and AD/CCT after both two procedures .  At 6 months, the UDVA and SE were −0.14 ± 0.06 and 0.33 ± 0.33D in FS-LASIK and −0.15 ± 0.06 and 0.35 ± 0.37D in Trans-PRK. There was no difference between the two groups in the postoperative UDVA and SE ( Both FS-LASIK and Trans-PRK caused the anterior corneal surface to become flatter, and the morphology of the corneal surface was irregular. Corneal HOAs were significantly increased after the two procedures. Trans-PRK using SPT introduced less corneal vertical coma than FS-LASIK. Corneal asphericity changes contributed to the corneal aberrations changes following FS-LASIK and Trans-PRK. Corneal refractive surgery has developed rapidly, and its safety, efficacy, and predictability have been continuously improved with the development of science and technology. Femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and small incision lenticule extraction (SMILE) are widely used in clinical practice due to their accuracy in making corneal flap and small incision, respectively. Transepithelial photorefractive keratectomy (Trans-PRK) has become a highly researched topic in recent years. It is characterized by one-step removal of the corneal epithelium and correction of refractive errors, which shortens the operation time and avoids the complications caused by FS-LASIK to make the corneal flap . With thIn principle, corneal refractive surgery aims to correct refractive errors by ablating a certain amount of corneal stroma in the central optical zone of the cornea. More central stromal tissue is ablated than peripheral tissue, which inevitably causes flattening of the cornea and causes changes in the shape of the anterior corneal surface inevitably. Many studies have shown that the corneal asphericity developed significantly from negative value to positive value before and after surgery –5, and tQ-value, aberrations) to a lesser extent than FS-LASIK. This study was conducted to compare the change patterns of corneal aberrations and corneal asphericity between FS-LASIK and Trans-PRK using SPT and to investigate how changes of corneal asphericity could account for the variation of corneal aberrations after the two procedures.Previous studies have found that the morphological changes of the anterior corneal surface after SMILE and FS-LASIK are different , and theThis prospective study included patients with low to moderate myopia, with or without astigmatism, who presented to the Tianjin Medical University Eye Hospital, Tianjin, China consecutivelyfrom July 2019 to January 2020. A nonrandomized controlled study design was used. Patients were divided into two groups: the case group for whom Trans-PRK was performed and the control group who received FS-LASIK. There were 44 patients (88 eyes) in the FS-LASIK group, including 23 males and 21 females. In the Trans-PRK group, there were 32 patients (64 eyes), including 15 males and 17 females. Inclusion criteria were as follows: age over 18 years with stable refraction for at least 2 years ; discontinuation of soft contact lens wear for a minimum of 2 weeks and rigid contact lens wear for at least 1 month prior to preoperative examination; myopia ≤−6.00D and astigmatism ≤ −2.00D; and no keratoconus tendency, no active eye disease, or systemic disease. Exclusion criteria were as follows: abnormal or keratoconic topography, active inflammation in the eyes, presence of periocular purulence, presence of serious ocular appendage lesions, history of previous ocular surgery, and presence of concurrent ocular diseases and systemic diseases that could affect corneal wound healing. The choice of surgical procedure primarily depended on the patients' preference (after a detailed description of the procedures). All patients signed informed consent.Q value and corneal aberration were measured using the Pentacam in a dark room. The patient was positioned and instructed to focus on automatic measurement immediately after blinking to obtain data so as to avoid interference caused by poor tear film quality and eyelid occlusion of the patient. The imaging quality result was shown as OK, and the corneal exposed area was greater than 9 mm. The Q values at diameters of 6, 7, 8, and 9 mm were measured on the anterior surface of the cornea. Corneal higher-order aberrations (HOAs), vertical coma (Z3−1), horizontal coma (Z31), and spherical aberration (Z40) were measured in a diameter range of 6 mm.All patients underwent routine preoperative complete eye examinations including measurement of the uncorrected (UDVA) and corrected (CDVA) distance visual acuity, manifest and cycloplegic refraction, slit-lamp evaluation of the anterior and posterior segment, intraocular pressure (IOP), axial length, keratometry, and corneal topography . The FS-LASIK and Trans-PRK were performed by the same skilled surgeon.μm. After all the bubbles under the corneal flap were absorbed and the flap was lifted, refractive ablation was performed with the Amaris 1050 excimer laser . The ORK-CAM software was used for surgical design, and the standard aspheric aberration-free mode was used. The surgeon asks the patient to look at the correct position during the ablation process. After the ablation, the eye was washed with a balanced salt solution and the corneal flap was repositioned. All patients were prescribed levofloxacin eye drops qid for 3 days, 0.1% fluoromethane drops qid for 4 weeks (reducing later to once a week), and 0.3% sodium hyaluronate drops qid for 3 months.In the FS-LASIK group, levofloxacin and 0.3% sodium hyaluronate eye drops were administered for 3 days before surgery, and 0.4% obuvacaine hydrochloride eye drops were used for surface anesthesia. The cornea flap was created with the IntraLase FS femtosecond laser system (USA). The flap hinge was located in the upper cornea. The flap diameter was set at 8.5 mm. According to the patient's own corneal thickness and ablation depth, the thickness of the corneal flap was set at a range of 95–110 μm in the center and 65 μm in the periphery in the 8 mm diameter range. The standard aspheric aberration-free mode of Trans-PRK using SPT is used for laser ablation of epithelium and stroma. Patients were requested to look at a pulsing green fixation light throughout the ablation. After laser ablation, the ocular surface was then thoroughly washed with cooled balanced salt solution, a bandage contact lens  was applied, and 0.3% tobramycin dexamethasone eye drops were instilled. All patients were instructed to use a topical instillation of levofloxacin, 0.1% fluoromethane, and sodium bromophenolate hydrate eye drops qid until removal of the contact lens. Following the healing of the corneal epithelium, we prescribed 0.1% fluoromethane drops qid for the first month (then reducing to once a month) and 0.3% sodium hyaluronate drops qid for 4 months.In the Trans-PRK group, levofloxacin and 0.3% sodium hyaluronate eye drops qid for 3 days were administered before surgery, prognac eye drops were administered 3 times (once every 15 min) before surgery to reduce the inflammatory response and pain, and 0.4% obuvacaine hydrochloride eye drops were used for surface anesthesia. All surgeries were performed with the Amaris 1050 excimer laser . The ORK-CAM software is used for surgical design. The epithelial thickness was set to 55 t test was used to compare the general data between the two groups. ΔQ, ΔHOA, ΔZ3−1, ΔZ31, and ΔZ40, respectively, represent the difference between each measurement value 6 months after operation and before operation. All corneal aberration results were statistically calculated using the root mean square (RMS) value. A repeated measurement two-way analysis of variance (ANOVA) was used to analyze the overall variation of parameters. The least-significant difference (LSD) was used for pairwise comparisons between and within groups. Pearson's correlation and simple regression were used to analyze the correlation. P values less than 0.05 were considered statistically significant.Data were analyzed using IBM SPSS version 26.0 . The normality of the data was verified with the Kolmogorov–Smirnov test. The independent sample P > 0 05). The detailed baseline characteristics by group are provided in There were no significant differences in any of the variables between the FS-LASIK and Trans-PRK groups  (P > 0.05) (The < 0.001) . Compare > 0.05) .Ftime = 218.324, P < 0.001). There was no significant difference in preoperative and postoperative ISV values between the two groups   .3−1, Z31, and Z40 in both groups after surgery as compared to the preoperative data are shown in 3−1, Z31, and Z40 for the both groups increased after surgery . There were no significant differences for the corneal HOAs, Z31, and Z40 between the two groups (P > 0.05). Only the Z3−1 in the Trans-PRK group was significantly lower than that of the FS-LASIK group (P < 0.001). Regarding the variation in the aberration, only Δ Z3−1 showed statistically significant difference between the two groups (P < 0.05), while Δ HOA, Δ Z31, and Δ Z40 showed no statistically significant differences between the two groups (P > 0.05)  .Q value was significantly correlated with the preoperative spherical equivalent (SE), ablation depth (AD), and the ratio of AD and CCT (AD/CCT) after both the FS-LASIK and Trans-PRK procedures    . The Δ Hctively) .In clinics, FS-LASIK and Trans-PRK are widely used in the correction of myopia. The safety and effectiveness of these two procedures have been widely verified –13. HoweQ value) at the anterior corneal surface was negative and increased from the center to the periphery. Our results showed that the Q value of the anterior corneal surface changed from negative to positive after FS-LASIK and Trans-PRK in the 6, 7, 8, and 9 mm zones. There was no statistically significant difference between the two groups. Nonetheless, we found that Q values after Trans-PRK surgery were lower than those of FS-LASIK at 1, 3, and 6 months. This may be the advantage of the Trans-PRK procedure. In addition, our results showed that ISV significantly increased after FS-LASIK and Trans-PRK. Although there was no significant difference between the two procedures, the value of ISV was lower at each follow-up time after Trans-PRK than that of FS-LASIK, which further indicated that Trans-PRK had certain advantages over FS-LASIK in maintaining the corneal surface regularity and retaining the morphology of the anterior surface. Regarding the changes of corneal morphology after surgery, the following reasons should be considered: First, the corneal epithelium undergoes a repair process after corneal refractive surgery, which can make the anterior surface of the cornea smooth. This is manifested as an uneven thickening of the corneal epithelium [The anterior corneal surface flattens gradually from the center to the periphery in physiological conditions. The corneal asphericity (ithelium , that isithelium . This maThe main sources of ocular aberrations are the cornea and lens including corneal aberrations and intraocular aberrations. Corneal refractive surgery was performed on the surface of the cornea, and the morphology of the anterior corneal surface changed significantly after surgery without affecting the lens. Therefore, corneal aberration was selected in the current study to evaluate the aberrations after refractive surgery. Zheng et al.  previousQ value for both FS-LASIK and Trans-PRK procedures. We also found that the increase in Q value was significantly correlated with the preoperative SE, AD, and AD/CCT after the two procedures. Zheng et al. [Q value, thus leading to an increase in HOAs of the cornea, especially spherical aberrations. Roberts and Dupps et al. [Another finding of our study was that the changes in corneal HOAs and spherical aberration were dramatically correlated with the increase in g et al.  believedg et al.  have shos et al. , 22 propIn summary, our study found that both FS-LASIK and Trans-PRK caused the anterior corneal surface to become flatter, and the morphology of the corneal surface to become irregular. The corneal higher-order aberrations were significantly increased after the two procedures. Trans-PRK using SPT introduced less corneal vertical coma than FS-LASIK. Corneal asphericity changes contributed to the corneal aberration changes following FS-LASIK and Trans-PRK. This should be considered when selecting patients for refractive surgery.


Purpose: Accumulating evidence indicates that N6-methyladenosine-related long non-coding RNAs (m6A-related lncRNAs) play a crucial role in the occurrence and development of several cancers. We aimed to explore the potential role of m6A-related lncRNA signatures in predicting prognosis for early-stage (stages I and II) colorectal cancer (CRC).Methods: m6A-related lncRNA data were obtained from The Cancer Genome Atlas. Univariate Cox regression analysis was used to screen for prognostic m6A-related lncRNAs. Immune characteristics were analyzed in different subgroups created via unsupervised clustering analysis. Next, patients were randomly divided into training and test cohorts. In the training cohort, least absolute shrinkage and selection operator (LASSO) regression was performed to establish a prognostic model. The predictive value of the signature was evaluated in the training and test cohorts. Drug sensitivity was also examined.Results: A total of 1,478 m6A-related lncRNAs were identified. Two subgroups were created based on the expression of seven prognostic m6A-related lncRNAs. Prognosis was worse for cluster 1 than for cluster 2, and cluster 1 was characterized by increased numbers of M2 macrophages, decreased numbers of memory B cells, and higher expression of checkpoint genes when compared with cluster 2. Five m6A-related lncRNAs were selected to establish a risk prediction signature via LASSO regression. The 3 years overall survival (OS) was higher in the low-risk group than in the high-risk group. The area under the curve at 1, 2, and 3 years was 0.929, 0.954, and 0.841 in the training cohort and 0.664, 0.760, and 0.754 in the test cohort, respectively. Multivariate Cox regression analysis suggests that the risk score was an independent predictor of OS in both the training and test cohorts. A prognostic nomogram based on the five m6A-related lncRNAs and their clinical features was built and verified. The high-risk group was more sensitive to chemotherapeutic drugs (camptothecin and cisplatin) than the low-risk group.Conclusion: We identified two molecular subgroups of early-stage CRC with unique immune features based on seven prognostic m6A-related lncRNAs. Subsequent analyses demonstrated the usefulness of a five m6A-related lncRNA signature as a potential indicator of prognosis in patients with early-stage CRC. Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies and is among the main causes of cancer-related deaths. Although the 5 years survival rate for early-stage CRC (stage I and stage II) can reach 90%, these patients are still at risk of recurrence, which is often fatal . WhetherN6-Methyladenosine (m6A)—which is present in messenger RNAs (mRNAs), non-coding RNAs (lncRNAs), and microRNAs (miRNAs) in most eukaryotes—is the most common epigenetic methylation modification of mammalian RNA . M6A modThe tumor microenvironment (TME) also plays an important role in tumor initiation and progression, and is comprised of tumor cells, stromal cells and innate and adaptive immune cells . ImmunotIn the present study, we identified two molecular subgroups of early-stage CRC with unique immune characteristics and showed the potential role of m6A-related lncRNA signatures in predicting prognosis in patients with early-stage CRC.This study was approved by the Medical Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (no. 2021ZSLYEC-006).https://portal.gdc.cancer. gov). According to the 8th edition of the American Joint Committee on Cancer, we identified patients with early-stage CRC. The clinical information included age, sex, TNM stage, and survival status. In addition, patients with no survival information or a survival time of less than 3 months were excluded from further evaluation to reduce statistical bias. To distinguish the mRNAs and lncRNAs, we downloaded the GTF files from Ensembl (http://asia.ensembl.org) for further analysis. Data were normalized, processed, and analyzed using R software 4.0.3.Initially, we downloaded RNA sequencing data  related to gene expression and the corresponding clinical information for CRC from The Cancer Genome Atlas (TCGA) website , demethylases (ALKBH5 and FTO), and recognition proteins   (p < 0.01).We extracted expression data for 23 m6A-related genes identified in previous studies, including methyltransferases (nd RBMX) . PearsonUnsupervised clustering analysis was used to classify patients with early-stage CRC into different subgroups based on the expression of the seven m6A-related lncRNAs associated with prognosis. The ConsensusClusterPlus package was used to perform the above steps and was repeated 1,000 times to ensure the stability of the classification . Then, wp-value <0.05 and a false discovery rate (FDR) q value <0.25 (GSEA (version 4.1.0) software was applied to determine the gene expression in cluster1 and cluster2 in the Molecular Signatures database (MSigDB) Collection (c2.cp.kegg. v7.2symbols.gmt) to further analyze the difference in KEGG pathway enrichment. The threshold of statistical significance was defined by a nominal ue <0.25 .p-value < 0.05.The ESTIMATE algorithm was used to calculate the ESTIMATE score, stromal score, and immune score between cluster 1 and cluster 2 . The CIBk: the number of m6A-related lncRNAs incorporated into the signature; βi: the coefficient for each m6A-related lncRNA; Si: the level of m6A-related lncRNA expression). Then, patients with early-stage CRC were divided into high-risk and low-risk groups based on the median score of the training cohort. The Kaplan–Meier method and log-rank test were used to evaluate the survival difference between the two risk groups in the training and test cohorts. The area under the curve (AUC) and time-dependent receiver operating characteristic (ROC) curves were examined to assess the predictive power of the signal for survival using the “survival” and “survivalROC” packages in R software. The “survcomp” package is used to calculate the Consistency index (C-index) to compare the prediction accuracy of prognostic features. Univariate and multivariate Cox regression analyses were conducted to verify the independence of the risk score by comparing common clinical features including age, sex, and stage. We used a forest map to demonstrate the results. The R package used in the above operation is “survival”, “pheatmap”, and “ggpupr”.First, patients with early-stage CRC were randomly divided into training and test cohorts at a ratio of 1:1 using the caret package. Second, least absolute shrinkage and selection operator (LASSO) Cox regression analysis was performed to analyze the best candidates and multiple m6A-related lncRNA characteristics for constructing the prognostic signature. Ten-fold cross-validation was used to prevent overfitting. Based on the above model, the risk score for each patient was calculated as follows: risk score = Nomograms are widely used to predict the outcomes of patients with cancer . Risk schttps://cancerrxgene.org) can be used for large-scale drug screening. Combined with genomic analysis, the response of tumors to chemotherapy drugs can be systematically identified. Using the GDSCdatabase, the half-maximal inhibitory concentration (IC50) of common chemotherapeutic drugs for gastrointestinal tumors  was calculated to evaluate the clinical application of this model in the treatment of early-stage CRC. The R package used in the above operation is “pRRophetic”. Then, we compared the difference in the IC50 between the high-risk group and low-risk group using the Wilcoxon signed-rank test. To visualize the data, box drawings were created using “pRRophetic” and “ggplot2” in R (The Cancer Drug Sensitivity Genomics (GDSC) database  was used for statistical analysis ,” “pRRophetic,” “caret,” “glmnet,” “timeROC,” “ggExtra”, “survcomp”). In the above statistical tests, a two-sided p < 0.05, The bioinformatic analysis of this study was performed as depicted in Unsupervised clustering identified two molecular subgroups as the optimal number of clusters, including 171 cases in cluster one and 80 cases in cluster two . Principp = 0.024), indicating that levels of immune cells differ based on molecular subgroup. Our analysis revealed enrichment of the proportion of M2 macrophages in cluster one, as well as a higher proportion of memory B cells in cluster two  and a test cohort (n = 124) in a 1:1 ratio. To build the m6A-related lncRNA signature for forecasting the prognosis of patients with early-stage CRC, LASSO Cox analysis was performed based on the seven m6A-related lncRNAs in the training cohort. The m6A-related lncRNA signature included five m6A-related lncRNAs and the coefficients of each  than low-risk patients, which may provide new treatment options for patients with early-stage CRC. Camptothecin is an alkaloid extracted from somerase . In agresomerase , camptitsomerase . It has somerase . More clin vivo and in vitro. Lastly, we did not explore the underlying mechanism of the studied m6A-related lncRNAs. Therefore, functional studies should examine these five lncRNAs individually and in combination to further verify the prediction accuracy of the signature and discover potential regulatory mechanisms. Despite these limitations, to the best of our knowledge, this is the first study to report an established external verification of m6A-related lncRNA signatures for early-stage CRC.However, our study had several limitations. First, this study was based on the TCGA database, and there was no external data set for verification. When searching for other data sets, such as the Gene Expression Omnibus, we were unable to locate one with both clinical information and the corresponding lncRNA and mRNA expression data. Future studies should use other data sets to verify the performance of our prognostic signature. Second, our findings must be verified In summary, our analysis of seven m6A-related lncRNAs associated with prognosis identified two molecular subgroups of early-stage CRC with unique immune features. Subsequent analyses demonstrated the usefulness of a five m6A-related lncRNA signature as a potential indicator of prognosis in patients with early-stage CRC. Further studies are required to explore the mechanisms underlying the association between the significant m6A-related lncRNAs and CRC prognosis and to verify the applicability of our prognostic signature.


However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC–MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).Hyperexpression of  However, both of these genes are not essential for viral replication and, thereby, can be deleted or replaced by foreign genes. Based on this principle, baculovirus is exploited as the vector for gene expression, and the baculovirus expression vector system (BEVS) is becoming one of the most important expression systems at present [Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vector as it can utilize silkworm as the host. However, compared with polyhedrin or p10, the expression levels of foreign genes are usually far lower. Hence, it is of great necessity and significance to deeply elucidate the mechanism underlying the hyperexpression of these two very late genes.Baculoviruses are the enveloped, double-stranded (ds) DNA viruses, which primarily infect the lepidopteran insects. They have been widely used as environmentally benign pesticides and recently developed for mammalian gene delivery . One of  present ,3. Thous present , some of present . Notably present , especiapolh and p10 promoters of BmNPV, and the results demonstrated that a majority of these proteins were common to both promoters. Among all the identified interacting proteins, actin-4 (Accession: S5M6C1) had the greatest number of unique peptides and was, therefore, utilized for further investigation. Here, we report that actin played an indispensable role in the hyperexpression of polh and p10.Generally, transcription represents the most important initial step in the regulation of gene expression, and transcriptional factors (trans-acting elements) also play important roles. This study aimed to identify proteins binding to Previous studies have shown that baculovirus is a prolific manipulator of actin. During the later stages of baculovirus infection, the expression of a subset of baculovirus early gene products induces the nuclear accumulation of monomeric actin (G-actin) and subsequent polymerization for filament (F-actin) formation . Both G-Alternatively, actin plays an integrating role in gene expression. It has been linked to numerous gene expression processes, ranging from gene activation to chromatin remodeling, maintenance of genomic integrity, and intranuclear movement of chromosomes and chromosomal loci. Actin interacts with all the transcribed genes , and is polh and p10 or has a global role in viral gene expression. This study aimed to elucidate the functional roles of actin in the transcription of viral very late genes.It remains unclear whether actin participates in the transcription of GGATCCATGTGCGACGAAGAAGTTGCC; the BamHI site is indicated by underline) and actin-R  and cloned into pIZ/V5-His plasmid using BamHI and EcoRI, generating pIZ-actin-Flag. Rabbit-HA  and Mouse-Flag  were used in the Co-IP assay.The BmN cell line was cultured in the SF900 II SFM  supplemented with 3% fetal bovine serum  at 27 °C. The T3 strain of BmNPV was termed as the wild-type (WT) virus. The expression plasmid pIZ-actin-Flag was generated from the pIZ/V5-His plasmid. Actin was amplified from cDNA (the cDNA was reverse transcribed from RNA derived from BmN cells) by using primers actin-F  and p10 fragment  were incubated with 100 g streptavidin-coated Dynabeads  in 400 µL binding buffer  for 30 min at room temperature under constant and slow rotation. A protein pull-down assay was conducted as described in a previous study [The probes were amplified from  primers . Biotin-us study . BmN cel6) were infected with BmNPV at an M.O.I of 10. At 15 h post infection (h p.i.), jasplakinolide  and cytochalasin D  were added, while the same amounts of DMSO were added in the control group.BmN cells . 95 °C for 5 min, then 40 cycles at 95 °C for 5 s, and 60 °C for 30 s were included in the reaction programs. Each sample was triplicated. The 2−ΔΔCt method was applied to calculate the relative expression levels of selected genes.BmN cells were harvested at the corresponding time points and the total RNA was extracted using the TRIzol™ reagent . Thereafter, the first-strand cDNAs was synthesized using 5 μg total RNA with the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix . The primers are listed in Multiple primer pairs are listed in Immunofluorescence was performed as described ,30. In sThe Co-IP assay was performed as described in a previous study . Respectpolh and p10 transcripts are abundant at 48 h p.i., we prepared biologically duplicate ChIP-seq libraries of actin from cells infected with vBm-actin-Flag. Chromatin was harvested, and immunoprecipitations were performed using Flag Tag antibody. In addition, no-Flag immunoprecipitation was used as a negative control. Briefly, 107 BmN cells infected with recombinant virus vBm-actin-Flag 48 h p.i. were collected and cross-linked with 1% formaldehyde for 10 min at 25 °C and then quenched by the addition of glycine to a final concentration of 125 mM. Afterward, samples were lysed on ice to obtain chromatins. Chromatins were sonicated to get soluble sheared chromatin (average DNA length of 200 to 500 base pairs (bp)). One part of the soluble chromatin was saved at −20 °C for input DNA, and the remainder was used for immunoprecipitation by Flag antibody.ChIP assays were performed as previously described, with few modifications ,33. ChIPB. mori genome using BWA. The same files were converted into bam format using SAMtools. For data scaling, ChIPseqSpikeInFree was used to normalize the ChIP-seq according to the spike-in control. MACS2 was adopted for peak calling using the default parameters. The ChIP-seq data generated in this study has been submitted to the NCBI BioProject database  under accession number PRJNA735791. In addition, the public ChIP-seq data generated from BmN cells were analyzed by the same method except for the spike process. The immunoprecipitated genomic DNA fragments were amplified using quantitative real-time PCR (qRT-PCR) with primers . For the data on each gene, triplicate biological samples and three technical replicates were used.Trimmomatic (version 0.38) was used to filter out low-quality reads. Clean reads were mapped to the reference  primers . qPCR wahttps://daehwankimlab.github.io/hisat2/, accessed on 1 December 2021, version: hisat 2-2.0.4) package, which initially removes a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. Genes that had two-fold changes (pIZ-actin-Flag group) between the control transcripts (pIZ/V5-His group) and whose p.adjust was less than 0.05 were identified as DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to identify the biological pathways altered in response to pIZ-actin-Flag transfection. The RNA-seq data have been submitted to the NCBI BioProject database  under accession number PRJNA735599.Cell transfection and co-transfection were conducted when the cell density reached 80%. BmN cells were transfected with pIZ-actin-Flag and pIZ/V5-His (as control), then were infected with BmNPV at 12 h post transfection (h p.t.) Total RNA from cells transfected with pIZ/V5-His and pIZ-actin-Flag was isolated and purified using TRIzol™ reagent at 48 h p.i. . RNA-seq libraries were prepared as described previously  with somp10 promoter was conducted using the firefly luciferase reporter. Luciferase was amplified from PGL4.11 by using primers Luc-F-1  and Luc-R-1  and cloned into pFastBacHTB using BamHI and EcoRI, generating pFast-polh-Luc. Luciferase was amplified from PGL4.11 by using primers Luc-F-2  and Luc-R-2  and cloned into pFastBacDual using XhoII and KpnI, generating pFast-p10-Luc. Construction of vBm-polh-luc and vBm-p10-luc was accomplished by transfer of the Tn7 cassette from the constructed pFast-polh-Luc or pFast-p10-Luc into the BmNPV bacmid. The primers are listed in The analysis of the polh and t test of GraphPad Prism 7.0 software. When p value was ≥0.05, differences were considered not significant (ns). When p value was <0.05, differences were considered significant. When p value was <0.01, differences were considered highly significant.Statistical differences were analyzed by Student’s polh and p10 promoter-interacting proteins, this study performed a DNA-protein pull-down assay. As shown in the workflow  were extracted, respectively, and incubated with biotinylated polh and p10 promoters to detect the promoter-binding proteins induced by virus infection. Three independent biological replicates were carried out, then distinct the bands in the BmNPV-infected group were selected and identified by on-bead trypsin digestion followed by LC-MS/MS.To identify a comprehensive set of workflow A, polh apolh and p10 were compared, which revealed a general trend that the more abundant proteins (the higher number of unique peptides identified) were common binding partners for each promoter. All these proteins were divided into two categories, including 10 viral proteins and 37 host proteins  or stabilized F-actin . First of all, we established the timing of nuclear recruitment and polymerization of actin, and found that F-actin began to appear in nuclei at 16 h post-infection (p.i.)  , which w8 h p.i. A,B. Howereatment C. These Considering that the transcription of RNA polymerase II is dependent on actin, a series of Co-IP assays were conducted to examine the potential association between actin and viral polymerase. In brief, BmN cells were co-infected with two types of viruses, including one expressing Flag-tagged actin and the other one expressing HA-tagged subunits of viral polymerase. BmN cells infected with only one virus were used to confirm the tag protein was expressed, which represented the input group. The four viral subunits of LEF-4, LEF-8, LEF-9, and p47 were found to encode components of viral RNA polymerase . At 72 hThe infected cells were treated with CD and Jas at 15 h p.i. and subjected to confocal microscopy at corresponding time points. At 20 h p.i., three subunits of viral polymerase were found to be translocated to the cytoplasm, and nuclear colocalization was attenuated C,F,I, depolh and p10 promoters, which is also a common binding motif for viral polymerase [To further elucidate the relationship between actin and the viral gene, ChIP-seq analysis was conducted and 54 actin-binding sites were identified in viral genome . As revelymerase . The strlymerase . As idenpolh and p10 promoter regions and the p10 coding region close to TSS (polh and p10 promoter regions were designed (polh and p10 promoter regions (F3) compared with the IgG control, respectively. Interestingly, the enrichment degree gradually increased from F1 to F3, and greater enrichment was observed sites closer to the TSS. The actin-binding specificity to the polh promoter (F3) was the highest among the four regions. It appeared that actin tended to be predominantly enriched in the promoter region of polh, but actin also occurred in the p10 gene body close to TSS (F4). Since actin lacks the intrinsic DNA-binding ability, the existing data suggest that actin indirectly binds to polh and p10 promoter regions by interacting with viral polymerase. Combined with the interactions between actin and viral polymerase, we concluded that actin was a component of polh and p10 TIC.ChIP-seq results were further confirmed by ChIP-qPCR analysis, which showed that actin was markedly enriched into the e to TSS D,E. The designed D,E for Pdesigned . Our resvlf-1), which stimulates expression from polh and p10 promoters [polh and p10 by binding to the burst sequence [Accumulating evidence suggests that transcription initiation is very complex and requires multiple factors to participate in the process. Given that actin dynamics is essential for viral polymerase recruitment, we wondered whether actin may function as a scaffold to organize TIC. To test this hypothesis, we sought to identify the relationships between actin and three key regulators IE1, PK1, and VLF-1. IE1 is highly conserved in all baculoviruses and considered as the major transcriptional trans-activator necessary for viral gene expression . PK1, a romoters . VLF-1 ssequence .As found from Co-IP assays, actin interacted and was colocalized with PK1, IE1, and VLF-1 C, therebpolh and p10 transcriptional levels were significantly upregulated at 48 h p.i.  analysis D,E. The polh and p10. Actin has no inherent DNA-binding capability, but it is enriched in the polh and p10 promoters via promoter-binding partners. The interactions of actin with components of viral polymerase TIC enable actin to serve as a scaffolding molecule to facilitate TIC formation (In this report, we discovered that actin played an important role in the hyperexpression of two very late genes, ormation . Recent polh and p10 and actin dynamics. According to our results, the disruption of actin dynamics by CD and Jas treatments delayed the transcription of polh and p10, indicating that the baculovirus-encoded polymerase was actin-sensitive (Firstly, we detected the direct correlations between the transcripts of ensitive . Co-IP aensitive . Accumulpolh and p10 further suggested actin as a component of viral polymerase TIC, as revealed by ChIP-seq and ChIP-qPCR results (polh and p10 promoters possess the conserved sequences essential for their expression. The viral very late promoters are transcribed by viral RNA polymerase and contain a TAAG sequence motif. TSS is always initiated at the second A in the TAAG motif. Moreover, three of the peak concentrations all showed an AAG motif (polh and p10 expression by binding to their promoters. Through interactions with TIC components, actin might exert its function as a scaffold protein for the assembly of the transcription complex. Moreover, other viral proteins that affected polh and p10 expression and host proteins not confirmed yet might be assembled or gathered together by the actin scaffold to participate in gene regulation.Secondly, the high enrichment of actin within the promoter sequences of  results E,F. It iAG motif D, clearlAG motif C. It waspolh and p10 (polh and p10 promoters. Thereafter, this study conducted an association analysis on RNA-seq and ChIP-seq data of actin-overexpression-induced host genes. GO and KEGG analyses indicated that baculovirus might hijack actin of the host transcription apparatus to facilitate its own transcription.Finally, we surprisingly found that overexpression of actin dramatically increased the transcript amounts of  and p10 A. The ad and p10 B,C. In tpolh and p10. The concentrations of transcription factors and co-factors close to the transcription initiation sites represent a sensitive limiting component that determines the number of transcripts produced. Overexpression of actin may enhance viral polymerase movement and then accelerate the recruitment of viral polymerase in the upstream transcription site, which facilitates TIC formation.In summary, our present findings supported the notion that actin positively regulated the expression of


MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group’s previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs’ proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle. As a native sheep bred in China, the Hu sheep (Ovis aries) has many excellent traits, such as good reproductive performance, high meat–bone ratio, and tender, juicy meat. As we know, the growth of muscle determines the yield of mutton . SkeletaMicroRNAs (miRNAs) are a kind of non-coding, single-stranded, small RNA sequence, which can be involved in post-transcriptional regulation, and which do not have the ability to encode proteins. As we know, miRNAs can target mRNA and degrade or suppress it, reducing mRNA level and translation efficiency . With thMiR-181a is a member of the miR-181 family, and the family is conserved among different species . The miRGenerally, miRNAs play a regulatory role by binding to the 3′UTR (untranslated region) of mRNA . So, we The purpose of our research was to understand the function of miR-181a on the proliferation and differentiation of SMSCs in Hu sheep. This study will contribute to the understanding of the function of miRNAs in Hu sheep skeletal muscle, and provides a theoretical basis for Hu sheep molecular breeding.All experimental schemes were implemented in accordance with the “Jiangsu Province laboratory animal management measures”, and approved by the Animal Ethics Committee of Yangzhou University .Sheep for tissue collection were provided by Suzhou Sheep Farm . After the ewe was anesthetized with 2% lidocaine hydrochloride , a 3-month-old Hu sheep fetus was collected by surgery; 2 g of skeletal muscle was then taken from the longissimus dorsi muscle.The complete medium used for HEK293T cells includes DMEM High-Glucose medium , 10% fetal bovine serum , and 1% penicillin-streptomycin-amphotericin .The SMSCs are separated according to the method previously described . When cu2.All cell culture conditions are 37 °C, 5% CO® Master qPCR Mix  was used to detect the expression levels of related genes in cells. A real-time fluorescence quantitative instrument  was used to perform QRT-PCR. Sheep GAPDH and U6 were used as reference genes. Each sample was performed in three repeated tests. The 2−ΔΔ CT method was used to calculate relative expression [Total RNA of SMSCs was extracted by Trizol  and stored in liquid nitrogen. We used the miRNA 1st strand cDNA Synthesis Kit (by stem-loop)  to synthesize miA cDNA, and we used miRNA Universal SYBR qPCR Master Mix  to detect the expression of microRNA-181a in cells. We used a one-step reverse transcription Kit  to synthesize gene cDNA, and the 2 × TSINGKEpression .Primers of miR-181a and U6 were designed using miRNA Design V1.01 software . U6 was used as a housekeeping gene for miRNA expression detection. Premier Primer 5.0 software  was used to design primers of related genes. GAPDH was used as a housekeeping gene for gene expression detection. These primers were synthesized by TsingKe . Primers are shown in MiR-181a mimic, mimic-NC (negative control), miR-181a inhibitor and inhibitor NC were designed and synthesized by RiboBio . Oligonucleotide sequences are shown in We used PCR to amplify the 3′UTR region of the YAP1 gene, which contains the predicted binding site of miR-181a and YAP1. Then, we used the homologous recombination method to clone the amplified fragment into a PMIR dual-luciferase reporter vector. The restriction sites are HindIII and mIuI. Finally, we confirmed the successful insertion of the target fragment by sequencing the constructed plasmid. In order to construct a mutant-type dual-luciferase reporter plasmid, we used the Fast Mutagenesis Kit V2  according to the instructions. Then, we sequenced the constructed mutant plasmid, and the result showed TTGGATG mutated to ACCTGCA. Primers are shown in The reagent used for cell transfection in this experiment is jetPRIME transfection reagent . In accordance with the instructions, we confirmed the transfection operations and reagent dosage.In the double luciferase experiment, HEK293T cells were used as tool cells. A 24-well cell culture dish was used to culture HEK293T cells. After the cells’ density reached 60%, we mixed the wild-type vector (PMIR-YAP1-3′UTR-WT) or the mutant vector (PMIR-YAP1-3′UTR-MT) with miR-181a mimic or NC, respectively. We then transfected the mixture into HEK293T cells. After 48 h of transfection, we used a dual-luciferase detection kit  to process the HEK293T cells. After the cells were processed, the luciferase activity was detected by a multi-mode micropore detection system .A 96-well cell culture dish was used to culture the SMSCs. After the cells’ density reached 40%, we transfected miR-181a mimic and mimic NC into the SMSCs. We used the CCK-8 Kit  to detect the absorbance of cells at 450 nm at 0 h (12 h after transfection), 24 h, 48 h, and 72 h. The absorbance-detection instrument is the multimode micropore detection system .A 24-well cell culture dish was used to culture the SMSCs. After the cells’ density reached 40%, we transfected miR-181a mimic and mimic NC into the SMSCs. After 48 h of transfection, we incubated the cells with EDU for two hours. Then, we used 1X PBS  to wash the cells. Next, we used 4% paraformaldehyde  to fix SMSCs for 0.5 h. Finally, we used EdU Apollo In Vitro Imaging Kit  to treat the SMSCs. A fluorescence inverted microscope  was used for cell image acquisition. Image-Pro software was used for data analysis. In each group of treatments, three areas were randomly selected to calculate the number of stained cells.A 6-well cell culture dish was used to culture SMSCs. After the cells’ density reached 40%, we transfected miR-181a mimic and mimic NC into the SMSCs. After 2 days of transfection, we collected the cells, added 70% ethanol, and fixed them overnight at 4 °C. Then, we used Cell Cycle Kit  to treat the SMSCs. Finally, an FACSAria SORP flow cytometer  was used to analyze cells. ModFit LT software was used for data analysis.® 594), Abcam, Cambridge, UK, 1:400) to incubate the SMSCs at 37 °C for 1 h. We then aspirated the primary antibody and washed the SMSCs with 1X PBST after the secondary antibody was incubated. DAPI  was used to stain the nuclei of the SMSCs. Finally, a fluorescence inverted microscope  was used to observe and photograph the SMSCs.A 12-well cell culture dish was used to culture SMSCs. The cell culture medium was the growth and differentiation medium. We transfected miR-181a mimic and mimic NC into the SMSCs after the cells reached 40% density. After 1 day of transfection, we replaced the growth medium with a differentiation medium. After 3 days of differentiation, we aspirated the medium and washed the SMSCs with PBS . Then, we fixed the SMSCs with 4% paraformaldehyde  for 30 min. Next, we used 0.5% Triton X-100  to permeate the SMSCs for 15 min. After 15 min of infiltrating the SMSCs, we used 1% BSA  to incubate the SMSCs for 60 min at 37 °C. Finally, we used the primary antibody  to incubate the SMSCs in darkness at 4 °C overnight. We then aspirated the primary antibody and washed the SMSCs with 1X PBST . Then, we used the secondary antibody  was used for relative expression of the protein obtained. The antibodies used in this study and their dilution ratios are as follows: Anti-CDK2 antibody , Anti-MYHC antibody , Anti-GAPDH antibody , Goat Anti-Rabbit IgG H&L(HRP) , Rabbit Anti-Mouse IgG H&L(HRP) .RIPA Cell Lysate  was used to lyse SMSCs. Then, we centrifuged the lysate cells at 12,000 rpm for 10 min and collected the supernatant. BCA protein quantification kit  was used to determine protein concentration. Next, we subjected the protein sample to polyacrylamide gel electrophoresis, and then transferred it to PVDF . We used 5% skimmed milk powder to seal the PVDF membrane, which was closed at 25 °C for 1.5–2 h. We then added the primary antibody and incubated it overnight at 4 °C. The next day, we used 1X TBST  to wash the PVDF membrane. After wash-over, we incubated cells with the secondary antibody at 25 °C for 1 h. We used Electrochemiluminescence (ECL) for imprinting display. The ChemDoct-test. We considered the data to be statistically significant only when p < 0.05. For each group of treatments, we performed 3 repetitions, and all data are presented as least-squares means ± SEM (standard error of the mean).Statistical analysis was performed by SPSS25.0 software . The comparative analysis of the two groups was performed by an unpaired Student’s To reveal the regulating effect of miR-181a on the proliferation of Hu sheep SMSCs, we transfected SMSCs with miR-181a mimic, mimic NC, miR-181a inhibitor, and inhibitor NC. Results showed that overexpression of miR-181a in SMSCs facilitates its expression, while interference results in the opposite effect a,b. ThisIn order to reveal the regulating function of miR-181a on SMSCs’ differentiation, we induced SMSCs to differentiate. We found that the content of miR-181a increased first and then decreased during the differentiation process a,b, whicTo further understand the molecular function of miR-181a in the process of proliferation and differentiation of SMSCs, first, we checked the mature sequence of miR-181a and found it is conserved in different species a. Then, In our study, we explored the function of miR-181a in the proliferation and differentiation of Hu sheep SMSCs. In the RNA-seq of prenatal and postpartum skeletal muscles of sheep, Liu et al. detected 1086 known miRNAs and 40 new miRNAs . The antAs we all know, miRNAs can bind to the target gene and regulate its expression . The ideIn conclusion, our research shows that MiR-181a suppresses the proliferation of SMSCs, facilitates the differentiation of SMSCs, and targets YAP1.


Nanotechnology has made an important contribution to oncology in recent years, especially for drug delivery. While many different nano-delivery systems have been suggested for cancer therapy, selenium nanoparticles (SeNPs) are particularly promising anticancer drug carriers as their core material offers interesting synergistic effects to cancer cells. Se compounds can exert cytotoxic effects by acting as pro-oxidants that alter cellular redox homeostasis, eventually leading to apoptosis induction in many kinds of cancer cells. Herein, we report on the design and synthesis of novel layer-by-layer Se-based nanocomplexes (LBL-Se-NCs) as carriers of small interfering RNA (siRNA) for combined gene silencing and apoptosis induction in cancer cells. The LBL-Se-NCs were prepared using a straightforward electrostatic assembly of siRNA and chitosan (CS) on the solid core of the SeNP. In this study, we started by investigating the colloidal stability and protection of the complexed siRNA. The results show that CS not only functioned as an anchoring layer for siRNA, but also provided colloidal stability for at least 20 days in different media when CS was applied as a third layer. The release study revealed that siRNA remained better associated with LBL-Se-NCs, with only a release of 35% after 7 days, as compared to CS-NCs with a siRNA release of 100% after 48 h, making the LBL nanocarrier an excellent candidate as an off-the-shelf formulation. When applied to H1299 cells, it was found that they can selectively induce around 32% apoptosis, while significantly less apoptosis (5.6%) was induced in NIH/3T3 normal cells. At the same time, they were capable of efficiently inducing siRNA downregulation (35%) without loss of activity 7 days post-synthesis. We conclude that LBL-Se-NCs are promising siRNA carriers with enhanced stability and with a dual mode of action against cancer cells. Nanotechnology has made important contributions to oncology in recent years, thanks to its uniquely appealing features for drug delivery, diagnosis, and imaging . The useNanomaterials, including polymers, lipids, inorganic carriers, polymeric hydrogels, and biomacromolecular scaffolds, have led to the development of systems that can deliver chemotherapeutics to tumor sites for achieving the intended therapeutic effect while minimizing unwanted side effects . Among tIt has been reported that SeNPs exhibit higher anticancer efficacy and fewer side effects compared to inorganic and organic selenium-containing molecular compounds . This haApart from the delivery of small molecular drugs, recent studies have confirmed the same synergistic effect of SeNPs in combination with siRNA for targeted gene silencing as well , 2020c. D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)  , is one ed unit) .in vitro uptake, cytotoxicity, and apoptotic effect on H1299 lung carcinoma cancer cells were investigated in comparison to siRNA-loaded nanocomplexes prepared from chitosan alone (CS-NCs). The selective anticancer effect of LBL-Se-NCs was investigated by including NIH/3T3 fibroblasts in the study as a normal cell line, similar to previous reports . SeNPs are first capped with chitosan during their synthesis to obtain Se@CS nanoparticles with excellent colloidal stability. Next, siRNA is electrostatically attached in the second step, forming Se@CS:siRNA nanoparticles. In the final step, the final layer of chitosan is applied (Se@CS:siRNA:CS) to provide protection to the siRNA and to create a net cationic surface charge for better cellular uptake . These l reports . Overall2SeO3 and chitosan  were purchased from Sigma-Aldrich . RPMI-1640, L-glutamine, Penicillin/Streptomycin solution  (P/S), Fetal Bovine Serum (FBS), Trypan Blue, 0.25% Trypsin-Ethylenediaminetetraacetic acid (EDTA), and Dulbecco’s phosphate-buffered saline (DPBS) were supplied by Gibco BRL . CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega . Hoechst 33342 was purchased from Molecular Probes . Lipofectamine RNAiMAX reagent was purchased from Invitrogen . jetPEI (jetPRIME®) was purchased from Polyplus-transfection® company (France).Na5′-CAAGCUGACCCUGAAGUUCtt-3′; antisense strand: GAACU UCAGGGUCAGCUUGtt-3′) and non-targeted siRNA  were purchased from Eurogentec . For the uptake experiments, the siCTRL duplex was labeled with Alexa Fluor 647 dye at the 5′ end of the sense strand (si-AF647) (Eurogentec). For the endosomal escape, red-labeled fluorescent oligonucleotides (AF647 ONs) were used (Eurogentec).Small interfering RNA against eGFP  chitosan solution under magnetic stirring at 900 RPM for half an hour at room temperature. The stabilized Se@CS nanoparticles were centrifuged at 21,000 g for 60 min allowing the collection of the Se@CS NPs while removing the remaining excess materials. The collected NPs were dispersed in RNase-free water. UV-Visible, dynamic light scattering (DLS)/Zeta potential, and Fourier transform infrared spectroscopy (FTIR) characterization were performed to confirm the synthesis of the core nanoparticles (Se@CS).Se nanoparticles stabilized with chitosan were prepared as previously reported with minor modifications . BrieflySe@CS:siRNA), and a third protective chitosan layer (Se@CS:siRNA:CS). These LBL-Se-NCs were synthesized in two steps, given as follows:The LBL-Se-NCs are composed of three layers: the core (Se@CS), a second layer with the siRNA (Surface loading of siRNA was carried on different weight ratios of siRNA to Se from 1:2.5 to 1:40 (w/w) under constant stirring at 400 RPM for 60 min.g/min for 60 min to remove all unreacted materials, after which the collected precipitate was resuspended in RNase-free water.Finally, after 60 min, the third layer was applied by adding a 0.5% (w/v) chitosan solution to the reaction under continuous stirring for another 60 min. The resulting NCs were centrifuged again at 21,000 g at a temperature of 4°C.In this study, chitosan is the most determining part with regard to siRNA complexation, cellular uptake, and endosomal release; so, we consider CS-NCs as a suitable control group to compare with the Se@CS:siRNA:CS group. In this regard, siRNA-chitosan nanocomplexes were prepared by the ionic gelation method, reported in a prior study, with some modifications . CS-NCs Hydrodynamic diameter and surface charge of LBL-Se-NCs were determined using a Zetasizer Nano  in each step of the synthesis.–1.The molecular structure of the core nanoparticles (Se@CS) was analyzed using FTIR by mixing the core nanoparticles with KBr  and pressing them into uniform pellets to perform scanning in the wavenumber range 4,000–400 cmTransmission electron microscopy (TEM) images were obtained at the VIB-UGent Transmission Electron Microscopy-Core facility using a JEM 1400 plus transmission electron microscope  operating at 80 kV. Samples were prepared for the TEM analysis by adding 50 μl of the nanoparticle dispersions on formvar/C-coated hexagonal copper grids (EMS G200H-Cu). After 20 min of waiting at room temperature, they were washed with double-distilled water several times.g, 60 min), the typical absorbance of nucleic acids at 260 nm was monitored. siRNA entrapment in NCs was further confirmed by agarose gel electrophoresis. A 2% agarose gel was prepared by dissolving agarose  in a 100 ml TBE buffer . GelRed  was added to the TBE buffer for siRNA detection and 5 μl of 5 × Gel Loading Buffer  was added for every 20 μl of the sample before running for 20 min at 120 V. A total volume of 20 μl was pipetted per lane and a siRNA ladder was added as well as a control. We used a Kodak digital science camera  to take an image of the gel under UV light .The entrapment of siRNA in NCs was assessed by a NanoDrop 2000c spectrophotometer . To determine the amount of free siRNA in the supernatant recovered after particle centrifugation  for 1 h and observing changes in the hydrodynamic diameter. The stability of the NCs was also evaluated by monitoring changes in the hydrodynamic diameter and zeta potential in RNase-free water at room temperature for both LBL-Se-NCs and CS-NCs over 20 days.in vitro release study was carried out by determining the released siRNA from LBL-Se-NCs and CS-NCs post-synthesis. In this technique, we separated the release media from the nanoparticles by centrifugation  were cultured in DMEM supplemented with 10% Calf serum, 2 mM L-Glutamine, and 100 μg/ml P/S in a humidified incubator at 37°C with 5% CO2 atmosphere. For all in vitro studies, NCs, prepared at a 1:40 weight ratio of siRNA to Se, were diluted in a culture medium to reach a final concentration of 2–64 nM siRNA.H1299 cells  stably expressing enhanced green fluorescence protein (eGFP) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 2 mM ® assay. On the next day, after replacing the cell culture medium with 100 μl of fresh medium, 100 μl of CellTiter-Glo® reagent was added in each well and mixed for 20 min on an orbital shaker  to induce cell lysis. The mix was further incubated at room temperature for 10 min to let the luminescent signal stabilize. Next, 100 μl suspension of each well was transferred to white 96-well plates and the luminescence signal was recorded by a GloMax® 96 Luminometer (Promega). Wells containing medium without cells were included to determine the background luminescence.The cells were seeded in 96-well plates at 7,500 cells per well (for 48 h study) and were allowed to attach overnight. On the next day, the cells were treated with the prepared NCs (2–64 nM siRNA concentration) for 4 h at 37°C in cell culture medium. Next, the cells were washed once with PBS and incubated in a fresh cell medium for an additional 24 h before they were prepared for measuring their metabolic activity by the CellTiter-Glo1). The cells were seeded in a 96 well culture plate and cultured for 24 h, after which they were treated with different concentrations of LBL-Se-NCs and CS-NCs (2–64 nM siRNA concentration) for 4 h at 37°C in a cell culture medium. Next, the cells were washed and incubated for an additional 24 h. On the next day, the culture medium was removed and transferred to another plate because the removed medium could contain dead cells that should also be included in the analysis. The cells were detached from the well plates using trypsin and then neutralized after 5 min with the medium that was kept from the previous step . Following centrifugation , the cell pellet was resuspended in staining buffer prepared from flow buffer  to which PI (1 μg/ml) and DiIC1 (10 nM) were added. The cells were incubated with the staining buffer for 30 min at 37°C before being analyzed with flow cytometry . The FlowJo software  was used to separate the cell population into three parts: Q1: live cells +/PI–); Q3: apoptotic cells ; and Q4: dead cells .Induction of apoptosis in NCs-treated H1299 and NIH/3T3 cells was evaluated by flow cytometry using propidium iodide (PI) and dihexaoxacarbocyanine iodide (DiICg), after which the cell pellet was resuspended in flow buffer for flow cytometry analysis.Uptake experiments were performed by seeding 9,000 H1299 and NIH/3T3 cells per well in 96-well plates which were allowed to attach overnight. The next day, NCs containing AF647 siRNA were prepared as described above. The cells were incubated with LBL-Se-NCs and CS-NCs (2–64 nM siRNA concentration) for 4 h at 37°C, after which they were washed and detached from the well plates using trypsin, and then neutralized with a complete cell culture medium. The cells were transferred to U-Bottom 96-well  plates and centrifuged  were measured for 60 s at a flow rate of 60 μl/min. Red fluorescence (excitation at 638 nm and detection with a 660/20 nm bandpass filter) was measured using a CytoFLEX flow cytometer. Finally, data analysis was performed using the FlowJo software for quantifying the percentage of positive cells (uptake [%]) as well as the relative mean fluorescence intensity per cell (rMFI) according to:For calculating the siRNA gene silencing efficiency on eGFP-H1299 cells, 7,500 cells per well were seeded (for 48 h study) in 96-well plates which were allowed to attach overnight. On the next day, the cells were treated with NCs containing eGFP-siRNA (sieGFP) and negative control-siRNA (siCTRL) (2–64 nM siRNA concentration) for 4 h at 37°C in RPMI. Next, the cells were washed and incubated for an additional 24 h. On the next day, following the same steps previously mentioned for flow cytometry, the cells were washed, detached, centrifuged, and resuspended in flow buffer for flow cytometry analysis. For each sample, the forward and side scatter  were measured for 60 s at a flow rate of 60 μl/min. The samples were excited with a 488 nm laser line and the signal was detected with a 525/40 filter using a CytoFLEX plate reader flow cytometer. The FlowJo software was used to measure eGFP expression according to the following equation:MFI siGFP indicates the mean fluorescence intensity of cells treated with GFP-siRNA and MFI siCTRL indicates the mean fluorescence intensity of cells treated with negative control-siRNA.2, 100% humidity, and 37°C). The 408 and 488 nm laser lines were used to excite the Hoechst-labeled nuclei and the eGFP protein, respectively.The eGFP knockdown efficacy was also evaluated by taking microscopy images after treatment with a Nikon A1R HD confocal laser scanning microscope , equipped with a laser box , detector box , and a 20X air objective lens  . The images were recorded using the NIS Elements software . Briefly, H1299-eGFP cells were seeded in 35 mm CELLview microscopy dishes with glass bottom  at a density of 75,000 cells per ml and were allowed to settle overnight. After removal of cell culture medium, the cells were treated for 4 h with LBL-Se-NCs, jetPEI, Lipofectamine, and CS-NCs, all containing 8 nM eGFP-siRNA and 8 nM naked eGFP-siRNA as a carrier-free control. Live-cell imaging was performed for evaluating knockdown efficacy after 24 h of treatment at 37°C. Before confocal imaging, the cells were washed and stained with Hoechst 33342 . After 20 min of incubation with Hoechst, the cells were washed three times with PBS. The stained cells were provided with a full cell culture medium and put on the microscope with an incubator to enable live-cell imaging at optimal environmental conditions  and incubated for 20 min 37°C and 5% CO2. Immediately after washing, the cells were provided with a full cell culture medium and were placed in the incubator of the microscope (37°C under a humidified 5% CO2 atmosphere).Endosomal escape was evaluated by a dequenching assay first published by For visualization, the 408, 488, and 633 nm laser lines were applied to excite the Hoechst-labeled nuclei, eGFP protein, and the AF647 ONs, respectively. Upon endosomal escape, free AF647 ONs would be released into the cytosol and accumulate into the nucleus. Data analysis with the ImageJ (FIJI)  softwaret-test was used for comparing conditions. All results are reported as mean ± SD and differences with a p-value < 0.05 considered significant.All statistical analyses were performed using the GraphPad software , and the propagation of errors was applied when necessary. One-way ANOVA or the Student’s –1 which correspond to the vibrational modes of chitosan, in particular, stretching vibrations of O-H and N-H. Compared to the spectrum of chitosan alone, there is a slight blue shift due to the formation of hydrogen bonds between selenium and chitosan (–1 (amide III). The peak located at 1,150 cm–1 is the result of chitosan deacetylation and is related to asymmetric vibrations of CO in the oxygen bridge. The absorption bands at 1,082 and 1,032 cm–1  are characteristic of its saccharide structure  for siRNA delivery in synergy with the native anticancer properties of selenium. For this, we started by synthesizing core SeNPs using chitosan as a coating (Se@CS). TEM imaging confirmed the formation of Se nanoparticles with a size of 74.3 ± 5 nm . The hydchitosan . Other ctructure . These vThe next step was to load siRNA on the Se@CS nanoparticles. siRNA was added at different weight ratios of siRNA to Se from 1:2.5 to 1:40 (w/w). After the addition of siRNA to the Se@CS core particles, the dispersion was centrifuged to remove any unbound siRNA. The presence of unbound siRNA in the supernatant was investigated by UV-Vis spectroscopy and agarose gel electrophoresis in order to determine the siRNA-loading capacity of the Se@CS core nanoparticles. When performing agarose gel electrophoresis on the supernatant, it was found that siRNA was fully complexed to the core nanoparticles at a ratio of 1:40, corresponding to an effective siRNA concentration of 160 nM . At highBased on these results, LBL-Se-NCs at a ratio of 1:40 (w/w) with an effective siRNA concentration of 160 nM were selected for further characterization. Upon coating with anionic siRNA, the positive surface charge of Se@CS decreased as expected, reaching a value of 35.0 ± 1.5 mV. At the same time, a decrease in the hydrodynamic diameter was observed from 218 ± 3 nm (Se@CS) to 174 ± 1.2 nm (Se@Cs:siRNA) . The reaThe process of layer coating on the core was also monitored by UV-Vis spectroscopy. As shown in We performed additional experiments in which the stability of Se@CS:siRNA nanocomplexes was examined after purification by centrifugation before and after applying the third chitosan layer. Analysis by DLS and simple visual inspection clearly showed that the presence of the third layer has an effect on the resuspension of the NCs in water. For nanocomplexes without the third layer, we could observe flocculation and sedimentation, while DLS also showed that the overall size of the NCs had increased with clear signs of aggregation .The TEM images were also recorded after the second (Se@CS:siRNA) and the third step (Se@CS:siRNA:CS). The addition of the second siRNA  did not On the other hand, the final chitosan layer was visible in the TEM images as a faint ‘halo,’ which resulted in an effective increase of the overall size of the nanoparticle to 95 ± 11 nm . Figure One of the first challenges for a drug delivery system is achieving good colloidal stability and not preventing aggregation . Therefog for 60 min to determine the amount of released siRNA in the supernatant. In the case of CS-NCs, more than 50% siRNA was already released after only 8 h, reaching 100% release after just 48 h. In contrast, the siRNA release from LBL-Se-NCs was only 35% after 7 days. These results show that, under equal experimental conditions, siRNA remains better associated to LBL-Se-NCs as compared to CS-NCs.Next, the siRNA release from LBL-Se-NCs and CS-NCs was monitored over time for a period of 7 days . LBL-Se-To determine the selective anticancer potential of LBL-Se-NCs, LBL-Se-NCs and CS-NCs were added to H1299 (cancer cells) and NIH/3T3  at concentrations ranging from 2 to 64 nM of siRNA, corresponding to 1–32 ppm of selenium. Note that untargeted siRNA was used in these experiments so as to judge the anticancer effect by the carriers alone. The CellTiter-Glo metabolic assay was used to quantify cell viability after 24 h. As shown in 1(5) fluorochrome and PI. DiIC1(5)  is a deep red, lipophilic dye that is selective for the mitochondria of live cells, causing high fluorescence intensity; meanwhile, for apoptotic cells, when the membrane potential of the mitochondria is no longer intact, the dye diffuses over the cytoplasm and the staining is less intense. PI , cannot cross intact plasma membrane and therefore will only be present in the DNA of necrotic cells where the plasma membrane has been compromised/permeabilized. Therefore, the combination of these two dyes allows for distinguishing apoptotic from necrotic cells by flow cytometry. Treatment with LBL-Se-NCs at a concentration of 8 nM resulted in 32.0 ± 0.7% and 5.6 ± 0.8% apoptosis in H1299 and NIH/3T3 cells, respectively . Furthermore, LBL-Se-NCs were found to selectively induce apoptosis in H1299 but not, or much less, in NIH/3T3 normal cells. Also, the uptake levels of LBL-Se-NCs in NIH/3T3 cells were remarkably lower than those in H1299 cells. Altogether, the selective anticancer effect of the LBL-Se-NCs in H1299 cancer cells could be confirmed, which can be directly linked to apoptosis induction in cancer cells by Se.In addition to this selective apoptosis, we evaluated the intracellular delivery of the developed LBL-Se-NCs in this study by evaluating the eGFP knockdown efficacy in H1299 cells. We found that LBL-Se-NCs induced similar knockdown levels as commercial transfection reagents like jetPEI and Lipofectamine, which confirms their high transfection efficiency. Interestingly, transfection efficiencies could be perfectly correlated with the endosomal escape efficiency of the various NCs . FurtherHerein, LBL-Se-NCs were successfully prepared using a simple layer-by-layer electrostatic assembly of siRNA and chitosan on Se@CS core nanoparticles. The first layer of chitosan functioned as an anchoring substrate for siRNA. A third and final CS layer was added as a protective layer, which provided outstanding colloidal stability for at least 20 d, which showed no variation in size or zeta potential irrespective of the suspension medium. Furthermore, the final CS layer ensured stable siRNA complexation with only 35% release 7 days post-synthesis, which was significantly better than CS-NCs. LBL-Se-NCs were found to selectively induce 32% apoptosis in H1299 cancer cells, which was 5.7 times more than that in NIH/3T3 normal cells. We could prove that this effect is linked to the presence of the Se core, since CS-NCs showed low levels of apoptosis in both cancerous and normal cells. LBL-Se-NCs were also found to be able to induce 35% downregulation of eGFP in H1299 cells, which was the case both just after synthesis and 7 days post-synthesis. This clearly showed the superior stability of the presented LBL nanocarriers herein, as all the other carriers  completely lost their efficacy over time.In conclusion, the induction of selective apoptosis and the improved siRNA silencing over extended periods of time make LBL-Se-NCs an excellent candidate as an off-the-shelf formulation with a dual mode of action against cancer cells.The original contributions presented in the study are included in the article/MS presented the initial idea for the research, planned the experiments of this study, and wrote most of the manuscript. MS and ES conducted most of the experiments including the synthesis and the characterization part. KB, JF, and RF-M supervised the experiment and advised on data analysis and writing. SD reviewed the data and the results. ZS advised on experiments and data analysis. HD provided the overall guidance and help for the use of confocal microscopy. RD performed the sample preparation for TEM and recorded the TEM images. All authors contributed to the article and agreed on the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wind is proposed as a mechanical energy source to flutter the dielectric film in wind-driven TENGs as it is clean, abundant, ubiquitous, and sustainable. Herein, we propose a TENG structure with dielectric films bent in four directions to collect the wind energy supply from all directions, unlike the conventional wind-driven TENGs which can only harvest the wind energy from one direction. Aluminum (Al) layer was intercalated within the dielectric film to improve electrostatic induction, resulting in improved triboelectric performances. Maximum open-circuit voltage (VThe online version contains supplementary material available at 10.1186/s40580-021-00276-5. With the advent of the fourth industrial revolution, the Internet of Things (IoT) technology that allows data to be sent and received in real time by attaching sensors to objects is developing rapidly –3. WearaCurrently, a variety of wireless sensors using IoT are ubiquitously utilized in military , industrTwo approaches for the mechanical energy harvesting, which can be generated by various human movements  and windInterest in mobility is growing in modern era; for example, drones with numerous wireless sensors are used in a wide variety of fields, including the observation of activities, video filming, and offline delivery –41. AccoHerein, we propose a wind-driven TENG structure with a cross-shaped dielectric film bent in four directions (C-TENG), which can produce a suitable external power supply in all wind directions, unlike the vehicles or subways that can run in only one direction. The TENG could efficiently harvest wind energy from all direction, suitable for drones flying in arbitrary directions. Additionally, another TENG, in which an Al layer is intercalated within a polytetrafluoroethylene (PTFE) film (CIA-TENG), was produced to improve electrostatic induction, resulting in improved triboelectric performances. Al and PTFE were selected as the triboelectric materials. The triboelectric performances of C-TENG and CIA-TENG were compared in this study. The effect of wind direction on the triboelectric performances was also tested, demonstrating that the wind energy can be harvested omnidirectionally.2. An Al foil  was used for the intercalated metal layer within the PTFE film. The CIA-film was produced by attaching the two PTFE tapes to both sides of the Al foil. The Al foil was protruded 5 mm from the PTFE film for the connection to the Al bottom electrode electrically. Dry etching was performed using a reactive ion echer  under following conditions; O2, CF4, and Ar gases , 20 mTorr, and 400 W for 60 s.A PTFE film tape  was used as a triboelectric material. The C-film was produced by sticking two PTFE film tapes each other and cutting them in the form of a cross-shape. The central square area of the C-film is 2 × 2 cm2.The Al tape  was used both top and bottom electrodes as well. After bending the cross-shaped CIA-film in four directions, the Al foil protruded from the CIA-film was electrically connected to the Al tape (attched to the bottom glass plate working as the bottom electrode) with a conductive carbon adhesive tape . Another Al tape (for the role of top electrode) was attached to the top glass plate with a size of 2 × 2 cmAn oscilloscope  and pre-amplifier  were used to measure the output voltage and current. During the measurement, wind was supplied with a commercial nitrogen blow gun, and the wind speed was measured using a commercial anemometer . The PTFE surface roughness was analyzed using AFM equipment .2  that actually touches the Al top electrode. Figure The C-TENG with the cross-shaped PTFE film (C-film) bent in four directions is depicted and shown in Fig. -1. In the case of the C-TENG, a maximum open-circuit voltage (Voc) of 153 V and short-circuit current (Isc) of 51.8 µA were measured  voltage was 2.36 mW at an external resistance of 1 MΩ in CIA-TENG  are the effective contact area of the dielectric film to the electrode, the effective dielectric thickness constant , triboelectric charges, and the separation distance depending on the time, respectively. In the open-circuit condition, the transferred charges (Q) between electrodes are 0; therefore, the Voc is affected by only σtribo and thus linearly proportional to only σtribo by Eq. (sc is given by: [v(t) is the fluttering speed of the dielectric film. According to Eq. , rendering a drop in Isc according to Eq.  bulbs in series  before and (b) after RIE etching. Fig. S2. (a) Maximum and RMS output voltage, and (b) average power vs. external resistance. Fig. S3. Voc and Isc of  the C-TENG and  the CIA-TENG using the unetched PTFE film at a wind speed of 9 m s-1. Fig. S4. Operating mechanisms of C-TENG;  initial electrification step and (c–f) repetitive triboelectric step. Fig. S5. Comparison of Isc at different (a) gaps and (b) wind speeds (interval: 0.05 s.Additional file 2: Movie S1. Vertical vibration of the CIA-TENG at a wind speed of 9 m s-1.Additional file 3: Movie S2. Demonstration of powering 25 LEDs by a CIA-TENG.


At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers  as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury. Spinal cord injury (SCI) is a devastating process leading to permanent neurological deficits for which there is currently no effective treatment. Injury, which comprises primary and secondary phases, provokes a cascade of events, that includes disruption of axons and blood vessels, primarily the damage to the blood–spinal cord barrier, extensive necrotic cell death, axon degeneration, demyelination, and the beginning of an immune response . These dCell transplantation therapies are one of the promising strategies for the treatment of SCI. Different types of cells are the focus of attention and research for example neural stem cells ,7,8, SchInflammation is a key goal of experimental and clinical treatment. MSCs play an important role in this process because it was found that MSCs can suppress inflammation ,24, downMSCs perform their regeneration activity not only through the cell replacement of injured cells but also via strong paracrine impact. MSCs have been found to secrete a broad range of trophic factors, bioactive molecules with neuroprotective and neuroregulatory effects ,30 to suPredicting the long-term outcome after SCI and evaluating the effectiveness of cell therapy is an important component of the treatment strategy. Biomarkers that can easily be repeatedly measured within the blood or cerebrospinal fluid (CSF) of the patients to determine progressive neurological condition would be highly beneficial, as it would allow rapid determination as to whether the patient was improving, worsening, or showed sustained neurological stability in response to their current treatment, thus providing a biological surrogate outcome measure. The rupture of the blood–brain barrier (BBB) and either damage to the neurons and glia of the spinal cord tracts results in the release of biomarkers, largely cellular components, which are specific in the indication of nervous tissue damage and include neurofilaments (NF), S100 calcium-binding protein β (S100β) and glial fibrillary acidic protein (GFAP). GFAP, the main intermediary filament of astrocytes, is found only in the CNS and is upregulated in reactive gliosis following CNS injury . GFAP isThe members of the S100 protein family are multifunctional proteins involved in the regulation of a wide range of cellular processes. One of the members of this large family is the S100B calcium-binding protein physiologically mainly produced by astrocytes in the CNS  and its There are three major subunits of neurofilaments based on molecular weight, namely heavy (NF-H), medium (NF-M), and light (NF-L). A major structural component of large motor axons is the phosphorylated neurofilament subunit NF-H (pNF-H). It can be detected in significant amounts in CSF or serum as a result of neuronal loss after injury . It is rIn our study, autologous bone marrow-derived MSCs (BM-MSCs) were isolated and expanded in vitro. After transplantation of BM-MSCs to a patient with SCI, the blood and CSF were analyzed for the presence of the pNF-H, S100 protein, and GFAP protein. Moreover, the dynamic changes to the chemokine/cytokine/growth factors’ profile in serum during the period of BM-MSCs treatment of a patient after SCI were examined . In our study, we have performed a descriptive analysis of the serum proteins’ level in the response to autologous BM-MSCs therapy of traumatic SCI. Cell transplantation treatment offers great potential to enhance the regeneration process after the injury. The blood and CSF of the BM-MSCs-treated patient were analyzed for the presence of the pNF-H, S100 protein, and GFAP protein. The chemokine/cytokine/growth factors’ profile was also observed in the serum during the follow-up period after the BM-MSCs treatment.The patient was a 35-year-old male who had a bicycle accident. He has a complete transverse lesion at the level of thoracic 6 (T6), completely lost the sensation and movements below T6 with no voluntary anal contraction and sensation to deep anal pressure. During 1-year follow-up, we did not find any clinically adverse reaction related to the BM-MSCs transplantation.The majority of cell therapy trials use autologous cells, specifically stem cells, but MSCs are dominant in registered clinical trials. The limiting and decisive factors in the use of MSCs in cell therapy of human SCI in clinical practice are mainly the type of MSCs—the origin from which the cells are isolated, doses, cell delivery method, and precise timing of cell administration after injury ,46. 2. BM-MSCs were expanded under standard in vitro conditions. The cultivation itself lasted 32 days and finally, 54 days after the injury (DaI), the cells were ready for application in an amount of 4.08 × 106/mL with 92.8% viability. In cell therapy trials the intrathecal cell delivery of MSCs predominates [5 to 40 × 107 [Twenty-two days after the injury, the patient´s BM was collected in an amount of 25 mL. Subsequently, according to standard procedures, BM-MSCs were isolated and placed in 15 culture flasks with an area of 75 cmominates . It was 40 × 107 . Althoug40 × 107 . In our 40 × 107 . Fortuna40 × 107 .Before application, MSC-specific markers were evaluated according to ISCT minimal definition criteria . Cells dIn contrast to a large number of reports on biomarkers in structural brain damage, only a few studies have investigated the role of biomarkers in patients with SCI. Quantitative evaluation of serum biomarkers could help to evaluate the course of damage and especially the success of tissue regeneration after the treatment. p < 0.001) higher than in the controls of healthy individuals , with a tendency to fluctuate slightly over the next forty days (p < 0.001) and GFAP (p < 0.01) was detected after the transplantation at 18 DaMSC. The serum level of S100 protein decreased almost 3-fold  and the level of GFAP protein decreased two and a half times  in comparison with theirs levels before BM-MSCs application at 48 DaI. In the following 6 months after BM-MSCs transplantation, there was a further decrease in glial protein levels with a tendency to approach the values of healthy individuals. Regarding the pNF-H protein, there were no significant changes in its level after BM-MSCs application during the follow-up period. After the cell transplantation, there was a decrease in pNF-H protein level during the follow-up period, but not to the extent that we found in glial proteins. The prolonged increase in plasma pNF-H in patients with central nervous system disorders may be caused due to continuous axonal degeneration, such as Wallerian degeneration or secondary damage of axons [In the first two weeks after the spinal cord injury (14 DaI), high levels of S100, GFAP, and pNF-H proteins were detected in the serum. The serum levels of all three proteins were significantly . Six months after BM-MSCs application, both cytokines retained significantly high serum levels with a tendency to increase over time when compared to control values. SCI caused an increase in interleukin IL-10 levels by almost 2-fold when compared to healthy controls . The production of IL-8 was significantly decreased during the first two weeks after the BM-MSCs therapy at 18 DaMSC (p < 0.001) in comparison with its levels before the BM-MSCs application at 14 DaI. However, with increasing time after BM-MSCs administration, IL-8 did not tend to increase its expression in the serum of a BM-MSCs treated patient.In contrast, interleukin IL-8 behaved in the opposite way to IL-10 and IL-1Ra cytokines C. IL-8 ip < 0.001, p < 0.001) (p < 0.001) in the level of IP-10 protein at 18 DaMSC in comparison with its level after injury at 14 DaI with a tendency to maintain a similarly low level within a six-month follow-up period after the cell administration. IP-10 is a small-inducible cytokine secreted during the inflammation by various cell types and acts as a potent inducer of apoptosis not only in infectious diseases but also during the development of the nervous system [p < 0.05) in comparison with its level after injury at 14 DaI. Based on our results, the RANTES protein tended to increase its level in response to BM-MSCs application. Similar results were obtained in other studies, where the expression of Ccl2/MCP-1 and Ccl5/RANTES was enhanced after MSCs transplantation and locomotor activity after spinal cord transection in mice was improved [Similarly, as we observed in cytokines levels, the interferon-gamma inducible protein of 10 kDa (IP-10), monocyte chemoattractant protein-1 (MCP-1) and Eotaxin display significantly high expression during 14 DaI with 10–20 times higher levels when compared to control values  D. Adminis system . IP-10 is system . IP-10 is system . Interess system . The sigimproved . In the improved . Eotaxinimproved ,77,78.In our study, we also monitored the changes in the expression of four growth factors in the serum , vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2), and platelet-derived growth factor (PDGF-AB/BB)) during the BM-MSCs treatment of SCI. p < 0.001) (p < 0.001). BM-MSCs application also caused an increase in FGF-2 and VEGF levels at 18 DaMSC, but not significantly. However, a significant increase in FGF-2 (p < 0.05) and VEGF (p < 0.01) occurred after a longer period after BM-MSCs application, at 180 DaMSC. VEGF is well-known as a specific mitogen promoting the growth and development of vascular endothelial cells. In combination with FGF-2 induces therapeutic angiogenesis [p < 0.001). However, after 6 months follow-up, the production of EGF protein tended to increase. SCI causes glial cells adjacent to the lesion to significantly alter their phenotypes and activities. EGF seems to play a decisive role in this process by conversion of astrocytes to their reactive form [Two weeks after the injury, we recorded significantly higher levels of PDGF-AB/BB, VEGF, and EGF in the patient’s serum compared with the control levels (< 0.001) . Adminisogenesis . Except,ogenesis ,80,81. Vogenesis . FGF-2 iogenesis . As we cogenesis . Our resive form . MSCs thive form .In summary, in this study, we compared the levels of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient before and after the treatment with autologous BM-MSCs. This study established two main points: (a) the effect of autologous BM-MSCs administration after SCI on the levels of cytokines/chemokines/growth factors  in the serum of an SCI patient, (b) the ability to detect the presence and the level status of SCI neuromarkers  in the serum and cerebrospinal fluid of an SCI patient treated with autologous BM-MSCs.Our preliminary results demonstrate that SCI, as well as subsequent BM-MSCs therapy, caused significant changes mainly in the levels of growth factors and neuromarkers detected in the serum. The most significant changes occurred in the serum levels of the neuromarkers GFAP, S100 as well as the growth factors FGF-2, PDGF-AB/BB, and VEGF when compared to the levels before and after the treatment.In summary, these findings demonstrate that neurodegenerative processes after traumatic SCI can be quantified biochemically by measuring S100, GFAP, and pNF-H levels in the serum or CSF. Together with inflammatory cytokines, growth factors and other biomolecules could emerge as surrogate markers and secondary outcome measures for neurodegenerative processes as well as for the rapid determination as to whether the patient was improving, worsening, or showed sustained neurological stability in response to the current treatment. Despite these very promising results, we acknowledge that the suitability of selected markers for SCI and the efficacy of BM-MSCs therapy after SCI have to be confirmed on a large group of patients in randomized clinical trials.A 35-year-old male with acute SCI and paraplegia after a bicycle accident, injured at the thoracic T6 level, was enrolled in our Department of Trauma Surgery. This study was approved by the institutional Ethics Committee of L. Pasteur University Hospital (06/EK/2020). Ethical guideline provisions from the Helsinki Declaration were followed. Written informed consent and the baseline status were obtained from all subjects before the study. v/v) Heparin anticoagulant  in the syringe to prevent coagulation. Directly after BM aspiration, all tubes were gently mixed and immediately (<1 h) transported on ice to the Associated Tissue Bank, Faculty of Medicine of P. J. Safarik University and L. Pasteur University Hospital. All cell manufacturing procedures were performed under strict sterile conditions. In the laboratory, BM aspirates were directly processed for the MSCs extraction using the red blood cell lysis method. Erythrocyte lysis buffer (10× stock solution: ultrapure water with 1.5 M ammonium chloride (NH4Cl), 100 mM potassium bicarbonate (KHCO3), 1 mM ethylenediaminetetraacetic acid (EDTA)  was added to the total volume of BM in the proportion 1:4 (BM volume: 1 x lysis buffer). The conical centrifugal tube was mixed manually for 10 min on ice and centrifuged at 200× g for 10 min at 4 °C. Promptly, after centrifugation, the supernatant was discarded and the pellet was resuspended in Dulbecco’s Modified Eagle’s Medium  and washed twice using centrifugation in the same conditions . Total cell number was determined by using a Bürker cell counting chamber. Finally, mononuclear cells were seeded at a density 1 × 106 cells/cm2 in 75 cm2 ventilated tissue culture treated flasks and cultivated in the complete cultivation medium containing alpha-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimycotic solution . After initial incubation for 2–3 days, the flasks were carefully washed to remove nonadherent cells and the medium was replaced with a fresh culture medium. The cultures were maintained in a humidified incubator at 37 °C with 5% CO2 and the medium was changed twice a week. The pathogen- and virus-free status was confirmed according to the safety analysis such as sterility, mycoplasma, and endotoxin detection before transplantation. In addition, tri-lineage differentiation tests in vitro and phenotypic control of BM-MSCs at 0 passages were used in this clinical study. The final product was prepared as 4.08 × 106 cell/dose of autologous BM-MSCs with 92.8% of viability. The cell viability was evaluated with trypan blue staining. The BM was collected in the operating room under the general narcosis. BM was aspirated aseptically from the iliac crest. BM aspirates were combined with a 5%  with 2% FBS , centrifuged at 300× g for 10 min, stained with a cocktail of antibodies CD 45 FITC, CD 90 FITC, CD 105 PE, CD 73 APC for 10 min in dark, washed with PBS containing 2% FBS and centrifuged at 300× g for 10 min. Cells were analyzed with a Becton Dickinson FACSCalibur using CellQuestPro software .Before application, immunophenotypic analysis of BM-MSCs was performed with a human MSC phenotyping kit . A minimum of 2 × 10g for 10 min at 4 °C. The total number of cells was counted after discarding the supernatant. Before application, the cell pellet was resuspended in sterile 0.9% saline solution in a final volume of 1 mL. For transportation, BM-MSCs in sterile normal saline were maintained in a temperature-controlled environment of approximately 4 °C under sterile and dark conditions. The stem cells suspension was transported as rapidly as possible. Two weeks before application, the FBS was changed to the patient’s autologous serum during cultivation. After reaching a confluence of above 90% , cells from passage 0 were washed twice with sterile PBS and treated with 0.25% Trypsin–EDTA solution  for 2 min at 37 °C. Cells solution was collected and centrifuged at 200× 6/mL) was performed by intrathecal injection in the operating theater under general anesthesia. The surgical wound was closed in a regular manner. The transplantation was performed using freshly harvested cells on 54 DaI.The transplantation of BM-MSCs (4.08 × 10g for 10 min. Serum samples were collected at given time points (n = 3). After centrifugation, human serum was aliquoted and stored at −80 °C until detection. Whole blood was collected in a covered test tube without anticoagulants and the clot was then removed by centrifuging at 1500× e points . Controlg for 10 min to remove any cells or large cellular fragments, aliquoted, and stored at −80 °C until detection.To minimize the number of invasive procedures to the injured patient, CSF was obtained during the same procedure as the BM-MSCs were administered intrathecally on 54 DaI, in the quantity of 5 mL. Collected CSF was separated by centrifugation at 300× ® Assays  to determine the mean of fluorescence intensity (MFI) values according to the manufacturer’s protocol and the MAGPIX Luminex platform. xPONENT software version 4.2 for MAGPIX  and Bio-Plex Manager 6.1  were used for data analysis. After creating a standard curve, concentrations were interpolated for each sample and expressed as pg/mL.Concentrations of 11 analytes  were quantified by a triplicate for each serum sample/time point using MILLIPLEXThe concentrations of target proteins in the serum and the CSF of the patient were detected using the Human S100, GFAP, and Phosphorylated Neurofilament H (pNF-H) Kits , following the manufacturer’s protocols. Samples were added into the wells of a polystyrene plate with pre-coated monoclonal antibodies against the specific protein of our interest and washed out with wash buffer after their respective additions to the wells. TMB substrate was used for coloration after the enzyme conjugate had already been thoroughly washed out of the wells. TMB reacted to form a blue product from the peroxidase activity and finally turned yellow after the addition of the stop solution. The optical density value of the target analyte in the sample was assessed at 450 nm using a microplate reader . Then, the standard curve was generated and the protein levels of tested samples were calculated according to the manufacturer’s guidance.p < 0.05 were considered statistically significant . The concentrations of proteins were expressed as the mean ± standard deviation (SD).Statistical differences among the groups were assessed by one-way ANOVA and Dunn’s multiple comparison test as post hoc analysis using GraphPad Prism software . Values